**2. Liposomes as model membranes to study the immunogenicity of lipids**

Liposomes can be formed by the modified [10] reverse phase evaporation method [32], where the cylindrical lipid phosphatidylcholine and the conical lipid phosphatidate are used in a 2:1 molar ratio. The higher proportion of cylindrical lipids allows the liposome to form a lipid bilayer association (smooth liposome) (**Figure 2A**). When the NPA inducers chlorpromazine (**Figure 2B**), promazine, procainamide or hydralazine are added, they interact with the conical lipids and generate a lipid rearrangement that forms an inverted micelle that is the center of the NPA (**Figure 2C**); therefore, a liposome bearing NPA is formed (**Figure 2D**). These drugs are used to treat completely different disorders, but they all cause as a side effect a disease similar to SLE [17]. These drugs are amphipathic, so they have a high affinity for lipid bilayers. When the drug is inserted into the liposome bilayer, it diffuses freely until it interacts with a phosphatidate molecule. This interaction is facilitated because the drugs have a positive charge and a triangular shape, while the phosphatidate has a conical shape and two negative charges (**Figure 1B** and **E**).

#### **2.1 Analysis of NPA formation on liposomes**

The formation of NPA on liposomes can be detected by flow cytometry [33]. In this technique, the laser beam dispersion by a liposome gives information regarding the liposome size and membrane complexity. The reverse phase evaporation method described above was designed to obtain unilamellar liposomes. Therefore, the size of the smooth liposomes is very similar to the size of the liposomes bearing NPA. With the use of other techniques like the thin-film hydration method that produce multilamellar liposomes, liposomes with more heterogeneous sizes are obtained. The membranes of smooth liposomes have less complexity than the membranes of liposomes bearing NPA. This difference in membrane complexity is revealed because the laser beam dispersion is higher in liposomes bearing NPA than in smooth liposomes [20, 34].

#### **2.2 Liposomes with drug-stabilized NPA induce a lupus-like disease in mice**

The development of this mouse model of lupus is based on the generation of an immune response against NPA that leads to the formation of anti-NPA antibodies. These antibodies bind to the NPA that are naturally present in mouse cells and cause their lysis [10, 33]. This model of lupus can be induced in three ways [10, 20, 35]:

1.Direct administration of the drugs chlorpromazine, promazine, hydralazine or procainamide to mice. In this case, the drugs stabilize NPA on mouse cell membranes.

*Anti-Non-Bilayer Phospholipid Arrangement Antibodies Trigger an Autoimmune Disease… DOI: http://dx.doi.org/10.5772/intechopen.106373*

#### **Figure 2.**

*Molecular structure of liposomes and NPA formation. Smooth liposomes made of phosphatidylcholine (blue polar head) and phosphatidate (green polar head) in a 2:1 molar ratio form a lipid bilayer (longitudinal view) (A). The drugs that induce NPA such as chlorpromazine, are amphipathic molecules with a triangular shape and a positive charge (B). When the NPA-inducer chlorpromazine (pink) is added to the smooth liposomes, it mainly interacts with phosphatidate, because this lipid has a conical shape with two negative charges. This interaction induces a lipid rearrangement that forms an inverted micelle that is the center of the NPA (C). In liposomes bearing NPA, most of the phosphatidate is forming inverted micelles inside NPA (longitudinal view) (D).*


The administration of liposomes bearing drug-stabilized NPA is the method that generates the highest titers of anti-NPA antibodies, particularly when chlorpromazine is used as the NPA inducer, because these NPA are larger and their epitopes are more exposed [20, 35, 36].

During the formation of NPA, phosphatidate molecules are shifted (as a result of their interaction with an NPA-inducing drug) from a bilayer to an inverted micelle, which lodges between the two lipid monolayers and forms the center of an NPA [33, 37]. The insertion of the micelle spreads the phospholipids polar heads that surround it and exposes new epitopes to the immune system (**Figure 3A**). This open spatial arrangement of phospholipids may favor the activation of the adaptive immune system cells, thereby leading to the formation of antibodies against the phospholipids that form the lipid bulge, which is structurally different from the surface of a normal lipid bilayer, where

#### **Figure 3.**

*Recognition of antigens by anti-NPA antibodies. Cross section of a liposome made of phosphatidylcholine (blue) and phosphatidate (green) bearing a chlorpromazine-induced NPA, which shows the spreading of the phosphatidylcholine molecules at the top of the NPA and the exposure of new antigens to the immune system (A). The immune system produces anti-NPA antibodies that recognize the polar heads of phosphatidylcholine (B). Anti-NPA antibodies bind to NPA on mouse cells and activate the complement cascade. Anaphylatoxins (C3a, C5a) are released to the medium, while opsonizing factors (C3b) bind to the cell; the membrane attack complex (MAC) is assembled and causes cell lysis.*

the polar heads are not separated [1]. Anti-NPA antibodies bind to the phospholipids that are spread at the top of the NPA (**Figure 3B**), which in liposomes is the lipid phosphatidylcholine, and not to the conical phospholipids or the inducers that form the inverted micelle (this micelle is submerged between the two monolayers of phospholipids). The specificity of the anti-NPA antibodies has been demonstrated with the use of haptens that represent the polar region of phospholipids; these studies confirmed that anti-NPA antibodies recognize the polar region of phosphatidylcholine [37].

Anti-NPA antibodies are the first antibodies that can be detected by ELISA or by flow cytometry in the serum of mice that received liposomes bearing NPA [10, 20, 35]. Most of these antibodies are IgG, and their affinity increase over time [36]. Six weeks after anti-NPA antibodies are detected, anti-histone and anti-cardiolipin antibodies can be detected by ELISA in the serum of these mice [20, 36, 37]. The delayed appearance of auto-antibodies against intracellular antigens can be explained if anti-NPA antibodies cause the lysis of NPA-bearing cells (perhaps by activating the complement cascade) (**Figure 3C**) and lead to the exposure of intracellular antigens, which now become targets of the immune system.

Mice that received NPA-stabilizing drugs, liposomes bearing drug-stabilized NPA or the H-308 monoclonal antibody developed a lupus-like disease characterized by piloerection, anorexia, weight loss, moderate alopecia and symmetrical facial lesions resembling the rash described in human lupus (**Figure 4**). The alopecic skin shows atrophic epidermis, diffuse lymphocytic infiltrates, an accentuated decrease

*Anti-Non-Bilayer Phospholipid Arrangement Antibodies Trigger an Autoimmune Disease… DOI: http://dx.doi.org/10.5772/intechopen.106373*

**Figure 4.**

*Pictures of mice that develop a lupus-like disease after the administration of liposomes bearing NPA. Representative photographs of 6-month-old female BALB/c mice that received liposomes bearing chlorpromazinestabilized NPA (mice are identified by marks made with a picric acid solution, which shows as yellow color). White arrows indicate the facial lesions. The green arrow indicates piloerection.*

of terminal hair follicles, inflammation around the matrical cells of hair bulbs, and widening of the external fibrous sheath with massive disaggregation of matrical cells [10, 20]. The kidneys show a mild enlargement of the mesangial matrix with thickened capillary walls in the glomeruli [20, 37]. Immune complex deposits were also found between the dermis and the epidermis (similar to the lupus band described in SLE patients), as well as in the capillaries and the renal mesangium [10].
